Mycobacterium tuberculosis infects one-third of the world population and kills more than 1.3 million people every year. Currently available Bacillus Calmette-Gu?rin (BCG) vaccine is insufficient in protecting against lung tuberculosis in adults and is not evidenced in controlling tuberculosis prevalence. Development of next-generation vaccine for tuberculosis is currently a global priority to combat millions of death caused by M. tuberculosis infections. Conventional CD4+ and CD8+ T cells are main targets for most vaccine candidates in present clinical trial pipeline. However, recent disappointing results from clinical trials in large human population suggested that a major gap remains for understanding the conserved pathways and new mechanisms for T cell activation, especially, CD8+ T cells. The surprising fact is that abundant CD8+ T cells recently discovered from M. tuberculosis-infected and healthy individuals were not conventional CD8+ T cells. Instead, these abundant CD8+ T cells are mucosal- associated invariant T (MAIT) cells that are responding to non-peptidic antigens presented by Major Histocompatibility Complex (MHC) class I like molecule MR1, as defined in our and other groups. Using an innate-like activation mechanism conserved in humans, MAIT cells are able to rapidly respond to infections, secret protective cytokines, and kill infected cells. Indeed, both in vitro and in vivo evidence supported the protective function of MAIT cells that inhibit the mycobacterial growth in infected cells and protect animals from severe infections. Therefore, MAIT cell population is advantageous to be considered as a target for developing anti-mycobacterial vaccine or therapy. Thus, what are antigens to activate MAIT cells in tuberculosis infection is a central question. We have found that MR1-presented antigens are non-peptidic and mycobacterial components activate MAIT cells. Together with our recently established metabolomic platform for antigen profiling, we will test the hypothesis that MR1 proteins present non-peptidic antigens to activate MAIT cells from tuberculosis patients. We propose here to use our structural and functional means to comprehensively profile the non-peptidic ligands associated with MR1 protein in M. tuberculosis infections and determine their structures and capability to activate MAIT cells in human tuberculosis. We will carry out two aims by (i) metabolomic profiling MAIT cell antigens from Mycobacterium spp. and recombinant MR1 proteins produced from mycobacterial-infected monocytic cells, (ii) determining the antigen-specific activation of MAIT cells from tuberculosis patients. If MAIT cell antigens are discovered and able to strongly activate MAIT cells against M. tuberculosis infections, these antigens can be provided as vaccination or therapeutic candidates for future animal studies and clinical evaluations.